Various analytical procedures and devices are commonly employed in flow-through assays to determine the presence and/or concentration of analytes that may be present in a test sample. For instance, immunoassays utilize mechanisms of the immune systems, wherein antibodies are produced in response to the presence of antigens that are pathogenic or foreign to the organisms. These antibodies and antigens, i.e., immunoreactants, are capable of binding with one another, thereby causing a highly specific reaction mechanism that may be used to determine the presence or concentration of that particular antigen in a biological sample.
There are several well-known immunoassay methods that use immunoreactants labeled with a detectable component so that the analyte may be detected analytically. For example, “sandwich-type” assay formats typically involve mixing the test sample with detection probes conjugated with a specific binding member (e.g., antibody) for the analyte to form complexes between the analyte and the conjugated probes. These complexes are then allowed to contact a receptive material (e.g., antibodies) immobilized within the detection zone. Binding occurs between the analyte/probe conjugate complexes and the immobilized receptive material, thereby localizing “sandwich” complexes that are detectable to indicate the presence of the analyte. This technique may be used to obtain quantitative or semi-quantitative results. Some examples of such sandwich-type assays are described in. by U.S. Pat. No. 4,168,146 to Grubb, et al. and U.S. Pat. No. 4,366,241 to Tom, et al. An alternative technique is the “competitive-type” assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are described in U.S. Pat. No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler, et al.
Despite the benefits achieved from these devices, many conventional lateral flow assays encounter significant inaccuracies when exposed to relatively high analyte concentrations. For example, when the analyte is present at high concentrations, a substantial portion of the analyte in the test sample may be left in excess and therefore not form complexes with the conjugated probes. Thus, upon reaching the detection zone, the uncomplexed analyte competes with the complexed analyte for binding sites. Because the uncomplexed analyte is not labeled with a probe, it cannot be detected. Consequently, if a significant number of the binding sites become occupied by the uncomplexed analyte, the assay may exhibit a “false negative.” This problem is commonly referred to as the “hook effect” or “prozone”.
Various techniques for reducing the “hook effect” in immunoassays have been proposed. For example, U.S. Pat. No. 6,183,972 to Kuo, et al. describes a strip of a porous material through which a test fluid suspected of containing the analyte can flow by capillarity. The strip has at least two distinct capture regions in which immobilized antibodies specific to a first epitope of the analyte are immobilized. Antibodies specific to a second epitope of the analyte are also employed that bear a detectable label. When present at sufficient concentrations, the analyte partially blocks binding of the immobilized antibody with the first epitope of the analyte. Thus, a sandwich of the immobilized antibody, analyte, and labeled antibody is formed in the capture regions. The emitted signal of the labeled antibody in each of the distinct capture regions thus provides a pattern of signals that is unique to the concentration of analyte. The set of signals is mathematically combined to create a monotonous dose-response curve to factor out the blocking of the binding between the immobilized antibody and the first epitope of the analyte.
A need still exists, however, for an improved technique of determining analyte concentration within the “hook effect” region in an accurate, yet simple and cost-effective manner.